- 1 – Buccal swab (0.5 mm punch)
- 2 – Hair (1 mm)
- 3 – Tooth (1 mm piece)
- 4 – Nail (1x2 mm)
- 5 – Saliva (0.5 µl)
- 6 – Amniotic fluic (1.0 µl)
- +/- – denote control reactions with and without purified DNA
Phusion DNA Polymerases
In a Thermo Scientific Phusion High-Fidelity DNA Polymerase, a DNA-binding domain is fused to a Pyrococcus-like proofreading polymerase. Due to this unique fusion technique, Phusion DNA polymerases generate PCR products with accuracy and speed unattainable with a single enzyme, even on the most difficult templates. In addition, Phusion DNA polymerases are tolerant of various PCR inhibitors. This allows robust amplification with minimal optimization. For hot-start PCR, Thermo Scientific Phusion Hot Start II High-Fidelity DNA Polymerase is an ideal choice allowing extreme specificity and improved robustness.
"After realizing that we could get the same number of cycles in roughly a quarter of the time (and at only slightly higher per unit cost), we changed exclusively to Phusion."
—Matt W. Ford, PhD student, Department of Biological Sciences, Idaho State University, USA.
"Different thermostable DNA polymerases were tested, but only Phusion polymerase, a Pyrococcus DNA polymerase-like enzyme fused with a double-stranded DNA-binding domain, had sufficient processivity."
—Hass M et al. (2008) J. Virol. 82:10207-10217.
"Earlier attempts with Turbo-Pfu polymerase were not successful. However, when using the highly processive Phusion polymerase instead, the usage of vector pHWSccdB led to positive clones."
—Stech J et al. (2008)
Nucleic Acids Research, 36:e139.
Features of Phusion DNA polymerases
In many applications such as cloning, site-directed mutagenesis or translating a DNA fragment into a protein, it is crucial to maintain the accurate DNA sequence during amplification. One incorrectly incorporated nucleotide may change the respective codon and result in the addition of an incorrect amino acid during translation. This, in turn, can affect folding and functional properties of the protein. On the other hand, deletion of a single nucleotide completely destroys the correct reading frame.
Phusion DNA Polymerases have extremely low error rates, thus setting a gold standard for Taq PCR. The error rate, determined by a modified lacI-based method , is approximately 50-fold lower than that of DNA polymerase and 6-fold lower than that of Pfu DNA polymerase (see Figure 1).
aError rate, number of errors (miscalled bases, inserted or deleted bases) divided by total number of bases.bDot, three successive negative flows during 454 sequencing. Figure reprinted with the permission from the corresponding author and the journal editor.
1. B.F. Frey & B. Suppmann (1995) Demonstration of the Expand PCR System's greater fidelity and higher yields with a lacI-based fidelity assay. Biochemica 2, 34-35.
2. Vandenbroucke et al. (2011) Minor variant detection in amplicons using 454 massive parallel pyrosequencing: experiences and considerations for successful applications. Biotechniques. 53:167-177.
3. Kinde et al. (2011) Detection and quantification of rare mutations with massively parallel sequencing. PNAS. 108(23):9530–9535.
Increased processivity allows short reaction times (extension 15-30 s/kb)
Phusion DNA polymerases incorporate more nucleotides per binding event as compared to other polymerases. This high processivity allows extremely short extension times and consequently reduced protocol times. Shortest protocol times can be achieved with Thermo Scientific Phusion Flash High-Fidelity PCR Master Mix, a product developed especially
for fast PCR.
Figure 4. Extreme speed and high yields with Phusion Flash. A 1.5 kb human cathepsin K gene was amplified with three different polymerases using varying extension times (10-60 s) with Thermo Scientific Piko Thermal Cycler. Only Phusion Flash was able to amplify the 1.5 kb gene with extremely short extension times of 10 and 20 s. It also produced superior yields of specific product compared to other enzymes tested.
Fewer reaction failures and minimal optimization
Phusion DNA polymerases are exceptionally tolerant of inhibitors and other challenging reaction conditions. This provides reliability by minimizing reaction failures, and even enables PCR from unpurified sample materials. Phusion DNA polymerases are the key components in Thermo Scientific Direct PCR Kits for human specimens and blood samples.
Figure 5. PCR from various human-derived samples without DNA purification. Different samples were placed directly into 20 μl PCR reactions. A 237 bp DNA fragment was successfully amplified using the control primers included in the Thermo Scientific Phusion Human Specimen Direct PCR Kit. Reactions were run in Thermo Scientific UTW reaction vessels using a Piko Thermal Cycler.
High product yields with minimal enzyme amounts
Due to the unique structure of the enzyme, Phusion DNA polymerases are also highly efficient. When compared to conventional polymerases, significantly fewer units of the enzyme are required for any PCR reaction. Speed and efficiency result in high product yields in minimal time. Also, these polymerases are highly robust which minimizes the need for reaction optimization.
Figure 6. Less enzyme - superior yield. A 3.8 kb fragment from human beta globin gene was amplified with threedifferent DNA polymerases. Phusion DNA Polymerase was able to amplify the 3.8 kb genomic fragment with a combined annealing and extensionstep of only 1 minute, thus being significantly faster than the two otherpolymerases tested. A single unit of Phusion DNA Polymerase produced higher yields than 2.5 or 5 units of the Pfu DNA polymerases.
Extreme specificity with the hot-start modification
The Affibody-based inactivation method of the Phusion Hot Start II DNA Polymerase greatly increases the specificity of PCR amplification. Both the DNA polymerase and the proofreading activities of Phusion Hot Start II DNA Polymerase are inactivated at room temperature by a reversibly binding Affibody ligand. This prevents nonspecific extension of the DNA template as well as degradation of the PCR primers during reaction setup. With Phusion Hot Start II DNA Polymerase, the reaction setup can be done at room temperature, enabling its use in high-throughput robotics. In addition to improved specificity, Phusion Hot Start II DNA Polymerase delivers improved robustness, allowing fewer reaction failures and minimizing the need for optimization.
Figure 7. Phusion Hot Start II DNA Polymerase provides extreme specificity and abundant yields. Five proofreading hot start DNA polymerases from major suppliers were used to amplify 1.7-2.3 kb fragments from human genomic DNA. Phusion Hot Start II DNA Polymerase provided high yields of specific products whereas all other enzymes delivered zero or low yields, with some also amplifying non-specific products.
Direct loading of PCR products on gels
Thermo Scientific Phusion Green format offers high convenience for the PCR setup and detection. This format is a combination of Phusion DNA Polymerase and Thermo Scientific 5X Phusion Green Buffer which includes a density reagent and two tracking dyes for direct loading of PCR products on gels. The Phusion Green Buffer does not interfere with the performance of Phusion DNA Polymerase and is compatible with downstream applications including DNA sequencing, ligation and restriction digestion.
Figure 8. 1X reaction mixture containing Phusion Green Buffer
A – unseparated in a well
B – blue and yellow dyes following electrophoresis
Thermo Scientific Phusion High-Fidelity DNA Polymerase
|Thermostable DNA polymerase with greatest accuracy, robust reactions, and high tolerance for inhibitors.||
|Thermo Scientific Phusion High-Fidelity PCR Master Mix with GC Buffer||Specially optimized PCR master mixes supplied with HF buffer for highest fidelity and GC buffer for amplification of GC-rich templates.||
|Thermo Scientific Phusion Flash High-Fidelity PCR Master Mix||PCR master mix enables the use of extremely short PCR protocols without compromising either the fidelity or the yield in the reaction.||
Thermo Scientific Phusion Hot Start II DNA Polymerase and PCR Master Mix
|Standalone enzymes and master mixes for improved PCR specificity and robustness.||
|Thermo Scientific Phusion Site-Directed Mutagenesis Kit||A versatile and efficient tool for introducing point mutations, insertions or deletions in any type of plasmid DNA.||
|Thermo Scientific Phusion U Hot Start DNA Polymerase and PCR Master Mix||Standalone enzymes and PCR mastermixes for high-fidelity amplification of uracil-containing templates and dUTP incorporation. Phusion U DNA Polymerase has a proprietary mutation in the dUTP binding pocket which allows it to tolerate uracil and incorporate dUTP during PCR.||
|Thermo Scientific Phusion U Multiplex PCR Master Mix||High performance Multiplex PCR Master Mix which enables simultaneous amplification of over 20 targets up to 2.5 kb. The master mix is based on Phusion U Hot Start DNA polymerases ensuring high yields of specific products.||
|Thermo Scientific Phusion buffers||Standard and detergent-free Phusion HF and GC buffers. Detergent-free versions are recommended for applications such as DHPLC and Microarray.||
For Research Use Only. Not for use in diagnostic procedures.